Diversity of β-Globin Mutations in Israeli Ethnic Groups Reflects Recent Historic Events

We characterized nearly 500 β-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. We found 28 different mutations in the β-globin gene, including three mutations (β^S, β^C, and β^O-Arab) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates—Druze and Samaritans—had a single mutation each. Fifteen of the β-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele—nonsense codon 37—appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of β-globin mutations can be largely explained by migration events that occurred in the past millennium.     

Alpha thalassemia and alpha-MRE haplotypes in Uruguayan patients with microcytosis and hypochromia without anemia

Alpha thalassemia is the most common genetic disorder across the world, being the α-^3.7 deletion the most frequent mutation. In order to analyze the spectrum and origin of alpha thalassemia mutations in Uruguay, we obtained a sample of 168 unrelated outpatients with normal hemoglobin levels with microcytosis and hypochromia from two cities: Montevideo and Salto. The presence of α-thalassemia mutations was investigated by gap-PCR, restriction endonucleases analysis and HBA2 and HBA1 genes sequencing, whereas the alpha-MRE haplotypes were investigated by sequencing. We found 55 individuals (32.7%) with α-thalassemia mutations, 51(30.4%) carrying the -α^3.7 deletion, one with the -α^4.2 deletion and three having the rare punctual mutation HBA2:c.-59C>T. Regarding alpha-MRE analysis, we observed a significant higher frequency of haplotype D, characteristic of African populations, in the sample with the -α^3.7 deletion. These results show that α-thalassemia mutations are an important determinant of microcytosis and hypochromia in Uruguayan patients with microcytosis and hypochromia without anemia, mainly due to the -α^3.7 deletion. The alpha-MRE haplotypes and the α-thalassemia mutations spectrum suggest a predominant, but not exclusive, African origin of these mutations in Uruguay.     

Factor-induced Reprogramming and Zinc Finger Nuclease-aided Gene Targeting Cause Different Genome Instability in β-Thalassemia Induced Pluripotent Stem Cells (iPSCs)

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However, it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps, we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in β-hemoglobin gene (HBB) that cause severe β-thalassemia (β-Thal), corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting, and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing, we uncovered seven copy number variations, five small insertions/deletions, and 64 single nucleotide variations (SNVs) in β-Thal iPSCs before the gene targeting step and found a single small copy number variation, 19 insertions/deletions, and 340 single nucleotide variations in the final gene-corrected β-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps, suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting.     

Frequency and spectrum of hemoglobinopathy mutations in a Uruguayan pediatric population

Hemoglobinopathies are the most common recessive diseases worldwide but their prevalence in Uruguay has not been investigated. In this study, 397 unrelated outpatient children from the Pereira Rosell Hospital Center (CHPR), as well as 31 selected patients with microcytic anemia and 28 β-thalassemia carriers were analyzed for hemoglobinopathies by using biochemical and molecular biology methods. Parametric and non-parametric methods were used to compare the hematological indices between groups of genotypes. Of the 397 patients in the first group, approximately 1% (0.76% HbS and 0.25% β-thalassemia) had a mutation in the HBB gene and 3.3% had β-thalassemia. These mutations had a heterogeneous distribution that varied according to individual ancestry. HbS was found exclusively in individuals with declared African ancestry and had a carrier frequency of 2.2%. The frequency of α-thalassemia carriers in outpatients of European and African ancestry was 1.2% and 6.5%, respectively. In contrast, the frequency of α-thalassemia carriers in patients with microcytic anemia was 25.8%, significantly higher (p < 0.01) than that observed in the sample as a whole and in Afro-descendants and Euro-descendants. Significant differences were observed in the hematological parameters between individuals with thalassemia genotypes and those with a normal genotype. These results indicate that hemoglobinopathies are a relevant health problem in Uruguay.     

Prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte,Brazil

α-Thalassemia, arising from a defect in α-globin chain synthesis, is often caused by deletions involving one or both of the α-genes on the same allele. With the aim of investigating the prevalence of α-thalassemia 3.7 kb deletion in the adult population of Rio Grande do Norte, 713 unrelated individuals, between 18 and 59 years-of-age, were analyzed. Red blood cell indices were electronically determined, and A₂ and F hemoglobins evaluated by HPLC. PCR was applied to the molecular investigation of α-thalassemia 3.7 kb deletion. Eighty (11.2%) of the 713 individuals investigated presented α-thalassemia, of which 79 (11.1%) were heterozygous (-α^3.7/αα) deletions and 1 (0.1%) homozygous (-α^3.7/-α^3.7). Ethnically, heterozygous deletions were higher (24.8%) in Afro-Brazilians. Comparison of hematological parameters between individuals with normal genotype and those with heterozygous α⁺-thalassemia showed a statistically significant difference in the number of erythrocytes (p < 0.001), MCV (p < 0.001), MCH (p < 0.001) and Hb A₂ (p = 0.007). This study is one of the first dedicated to investigating α-thalassemia 3.7 kb deletion in the population of the State Rio Grande do Norte state. Results obtained demonstrate the importance of investigating this condition in order to elucidate the causes of microcytosis and hypochromia.     

Pressure from TRIM5α Contributes to Control of HIV-1 Replication by Individuals Expressing Protective HLA-B Alleles

The expression of certain HLA class I alleles, including HLA-B*27 and HLA-B*57, is associated with better control of human immunodeficiency virus type 1 (HIV-1) infection, but the mechanisms responsible are not fully understood. We sought evidence that pressure from the human restriction factor TRIM5α (hTRIM5α) could contribute to viral control. The hTRIM5α sensitivity of viruses from both HLA-B*57-positive (HLA-B*57⁺) and HLA-B*27⁺ patients who spontaneously controlled viral replication, but not viruses from viremic patients expressing these alleles, was significantly greater than that of viruses from patients not expressing these protective HLA-B alleles. Overall, a significant negative correlation between hTRIM5α sensitivity and viral load was observed. In HLA-B*57⁺ patients, the T242N mutation in the HLA-B*57-restricted TW10 CD8⁺ T lymphocyte (CTL) epitope was strongly associated with hTRIM5α sensitivity. In HLA-B*27⁺ controllers, hTRIM5α sensitivity was associated with a significant reduction in emergence of key CTL mutations. In several patients, viral evolution to avoid hTRIM5α sensitivity was observed but could be associated with reduced viral replicative capacity. Thus, in individuals expressing protective HLA-B alleles, the combined pressures exerted by CTL, hTRIM5α, and capsid structural constraints can prevent viral escape both by impeding the selection of necessary resistance/compensatory mutations and forcing the selection of escape mutations that increase hTRIM5α sensitivity or impair viral replicative capacity.     

Spectrin Tetramer Formation Is Not Required for Viable Development in Drosophila

The dominant paradigm for spectrin function is that (αβ)₂-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and β_H-chains with high affinity, resembling other non-erythroid spectrins. However, α-spec^R22S, a tetramerization site mutant homologous to the pathological α-spec^R28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to β_H-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrin^R22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.     

NF-κB Activation Coordinated by IKKβ and IKKε Enables Latent Infection of Kaposi's Sarcoma-Associated Herpesvirus

All herpesviruses share a remarkable propensity to establish latent infection. Human Kaposi''s sarcoma-associated herpesvirus (KSHV) effectively enters latency after de novo infection, suggesting that KSHV has evolved with strategies to facilitate latent infection. NF-κB activation is imperative for latent infection of gammaherpesviruses. However, how NF-κB is activated during de novo herpesvirus infection is not fully understood. Here, we report that KSHV infection activates the inhibitor of κB kinase β (IKKβ) and the IKK-related kinase epsilon (IKKε) to enable host NF-κB activation and KSHV latent infection. Specifically, KSHV infection activated IKKβ and IKKε that were crucial for latent infection. Knockdown of IKKβ and IKKε caused aberrant lytic gene expression and impaired KSHV latent infection. Biochemical and genetic experiments identified RelA as a key player downstream of IKKβ and IKKε. Remarkably, IKKβ and IKKε were essential for phosphorylation of S⁵³⁶ and S⁴⁶⁸ of RelA, respectively. Phosphorylation of RelA S⁵³⁶ was required for phosphorylation of S⁴⁶⁸, which activated NF-κB and promoted KSHV latent infection. Expression of the phosphorylation-resistant RelA S⁵³⁶A increased KSHV lytic gene expression and impaired latent infection. Our findings uncover a scheme wherein NF-κB activation is coordinated by IKKβ and IKKε, which sequentially phosphorylate RelA in a site-specific manner to enable latent infection after KSHV de novo infection.     

A Truncated Form of Rod Photoreceptor PDE6 β-Subunit Causes Autosomal Dominant Congenital Stationary Night Blindness by Interfering with the Inhibitory Activity of the γ-Subunit

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.     

The Movement Ecology of the Straw-Colored Fruit Bat,Eidolon helvum,in Sub-Saharan Africa Assessed by Stable Isotope Ratios

Flying foxes (Pteropodidae) are key seed dispersers on the African continent, yet their migratory behavior is largely unknown. Here, we studied the movement ecology of the straw-colored fruit bat, Eidolon helvum, and other fruit bats by analyzing stable isotope ratios in fur collected from museum specimens. In a triple-isotope approach based on samples of two ecologically similar non-migratory pteropodids, we first confirmed that a stable isotope approach is capable of delineating between geographically distinct locations in Sub-Saharan Africa. A discriminant function analysis assigned 84% of individuals correctly to their capture site. Further, we assessed how well hydrogen stable isotope ratios (δ²H) of fur keratin collected from non-migratory species (n = 191 individuals) records variation in δ²H of precipitation water in sub-Saharan Africa. Overall, we found positive, negative and no correlations within the six studied species. We then developed a reduced major axis regression equation based on individual data of non-migratory species to predict where potentially migratory E. helvum (n = 88) would come from based on their keratin δ²H. Across non-migratory species, δ²H of keratin and local water correlated positively. Based on the isoscape origin model, 22% of E. helvum were migratory, i.e. individuals had migrated over at least 250 km prior to their capture. Migratory individuals came from locations at a median distance of about 860 km from the collection site, four even from distances of at least 2,000 km. Ground-truthing of our isoscape origin model based on keratin δ²H of extant E. helvum (n = 76) supported a high predictive power of assigning the provenance of African flying foxes. Our study highlights that stable isotope ratios can be used to explain the migratory behavior of flying foxes, even on the isotopically relatively homogenous African continent, and with material collected by museums many decades or more than a century ago.     

Genetic Heterogeneity of the β-Globin Gene in Various Geographic Populations of Yunnan in Southwestern China

Objectives

The aim of this study was to investigate the geographic distribution of β-globin gene mutations in different ethnic groups in Yunnan province.

Methods

From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes.

Results

Eighteen β-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of β-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT) was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T) was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A) was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T) was mainly linked to haplotype VII (+ - - - - - +) and IVS-II-654 (C>T) was only linked to haplotype I (+ - - - - + +).

Conclusion

Our data underline the heterogeneity of β-globin gene mutations in Yunnan. This distribution of β-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.     

Identification and Characterization of a Mucilaginibacter sp. Strain QM49 β-Glucosidase and Its Use in the Production of the Pharmaceutically Active Minor Ginsenosides (S)-Rh1 and (S)-Rg2

Here, we isolated and characterized a new ginsenoside-transforming β-glucosidase (BglQM) from Mucilaginibacter sp. strain QM49 that shows biotransformation activity for various major ginsenosides. The gene responsible for this activity, bglQM, consists of 2,346 bp and is predicted to encode 781 amino acid residues. This enzyme has a molecular mass of 85.6 kDa. Sequence analysis of BglQM revealed that it could be classified into glycoside hydrolase family 3. The enzyme was overexpressed in Escherichia coli BL21(DE3) using a maltose binding protein (MBP)-fused pMAL-c2x vector system containing the tobacco etch virus (TEV) proteolytic cleavage site. Overexpressed recombinant BglQM could efficiently transform the protopanaxatriol-type ginsenosides Re and Rg₁ into (S)-Rg₂ and (S)-Rh₁, respectively, by hydrolyzing one glucose moiety attached to the C-20 position at pH 8.0 and 30°C. The K_m values for p-nitrophenyl-β-d-glucopyranoside, Re, and Rg₁ were 37.0 ± 0.4 μM and 3.22 ± 0.15 and 1.48 ± 0.09 mM, respectively, and the V_max values were 33.4 ± 0.6 μmol min⁻¹ mg⁻¹ of protein and 19.2 ± 0.2 and 28.8 ± 0.27 nmol min⁻¹ mg⁻¹ of protein, respectively. A crude protopanaxatriol-type ginsenoside mixture (PPTGM) was treated with BglQM, followed by silica column purification, to produce (S)-Rh₁ and (S)-Rg₂ at chromatographic purities of 98% ± 0.5% and 97% ± 1.2%, respectively. This is the first report of gram-scale production of (S)-Rh₁ and (S)-Rg₂ from PPTGM using a novel ginsenoside-transforming β-glucosidase of glycoside hydrolase family 3.     

Key Residues in the Nicotinic Acetylcholine Receptor β2 Subunit Contribute to α-Conotoxin LvIA Binding

α-Conotoxin LvIA (α-CTx LvIA) is a small peptide from the venom of the carnivorous marine gastropod Conus lividus and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. It can distinguish the α3β2 nAChR subtype from the α6β2* (* indicates the other subunit) and α3β4 nAChR subtypes. In this study, we performed mutational studies to assess the influence of residues of the β2 subunit versus those of the β4 subunit on the binding of α-CTx LvIA. Although two β2 mutations, α3β2[F119Q] and α3β2[T59K], strongly enhanced the affinity of LvIA, the β2 mutation α3β2[V111I] substantially reduced the binding of LvIA. Increased activity of LvIA was also observed when the β2-T59L mutant was combined with the α3 subunit. There were no significant difference in inhibition of α3β2[T59I], α3β2[Q34A], and α3β2[K79A] nAChRs when compared with wild-type α3β2 nAChR. α-CTx LvIA displayed slower off-rate kinetics at α3β2[F119Q] and α3β2[T59K] than at the wild-type receptor, with the latter mutant having the most pronounced effect. Taken together, these data provide evidence that the β2 subunit contributes to α-CTx LvIA binding and selectivity. The results demonstrate that Val¹¹¹ is critical and facilitates LvIA binding; this position has not previously been identified as important to binding of other 4/7 framework α-conotoxins. Thr⁵⁹ and Phe¹¹⁹ of the β2 subunit appear to interfere with LvIA binding, and their replacement by the corresponding residues of the β4 subunit leads to increased affinity.     

High Resolution Melting Analysis: A Rapid Screening and Typing Tool for Common β-Thalassemia Mutation in Chinese Population

β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies.     

Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α⁻β⁻ spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and α⁻β⁻ spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α⁻β⁻ spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α⁻β⁻ spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.     

Distinctive Patterns of Evolution of the δ-Globin Gene (HBD) in Primates

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψβ(HBBP1), and the late expressed genes, δ (HBD) and β (HBB). While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.     

MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8⁺ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations.     

Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated α-synuclein (αS^D) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T αS and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of αS^D (disease-associated αS) in these mice, an ELISA was designed to specifically quantify αS^D in sick mice. Analysis of the central nervous system in this mouse model showed the presence of αS^D mainly in the caudal brain regions and the spinal cord. There were no differences in αS^D distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of αS^D immunoreactivity using an antibody against Ser129 phosphorylated αS by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of αS. The propagation of αS^D, suggesting the involvement of a “prion-like” mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.